Change In Quantal Size Research Articles

Endogenous NMDA-receptor activation regulates glutamate release in cultured spinal neurons.

N-methyl--aspartate (NMDA) receptor activation plays a fundamental role in the genesis of electrical activity of immature neurons and may participate in activity-dependent aspects of CNS development. A recent study has suggested that NMDA-receptor-mediated glutamatergic neurotransmission might occur in the developing spinal cord via activation of nonsynaptic receptors, but the details of NMDA-receptor activation in the developing CNS are not yet well understood. We describe here a model of cultured spinal neurons that display ongoing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor activity characterized by spontaneous excitatory postsynaptic currents (EPSCs), with NMDA-receptor activity detectable only as single channel events. -2-amino-5-phosphonovaleric acid (100 microM) and tetrodotoxin (TTX) 100 nM each reduced the occurrence of spontaneous AMPA EPSCs; quantal analysis showed a decrease in the number of released quanta but no changes in quantal size, indicating that NMDA-receptor activation and Na+ channel activity affect the generation of spontaneous AMPA EPSCs, at least in part, via mechanisms that impinge on the presynaptic terminal. Once the Mg2+-block was released, activity of NMDA receptors dramatically increased the release of quantal and multiquantal amounts of glutamate, indicating that the NMDA receptors are physiologically coupled to glutamate release. In Mg2+-free solution, TTX application elicited an increase in the number of quantal AMPA EPSCs and a reduction in the number of multiquantal EPSCs, consistent with an effect of NMDA-receptor activation on presynaptic terminals. Our results suggest that endogenous activity at a small number of NMDA receptors can regulate the release of neurotransmitters at developing AMPA synapses.

NMDA Receptor-Dependent and Metabotropic Glutamate Receptor-Dependent Forms of Long-Term Depression Coexist in CA1 Hippocampal Pyramidal Cells

We have found that two distinct forms of long-term depression (LTD), one dependent on the activation of NMDA receptors (NMDARs) and the other dependent on the activation of metabotropic glutamate receptors (mGluRs), coexist in pyramidal cells of the CA1 region of the hippocampus of juvenile rats (11–35 days). Both forms were pathway specific, required membrane depolarization, and were blocked by chelating postsynaptic Ca2+with BAPTA. The mGluR-LTD, but not the NMDAR-LTD, was blocked by the T-type Ca2+channel blocker Ni2+and intracellular administration of a protein kinase C inhibitory peptide. In contrast, the protein phosphatase inhibitor Microcystin LR blocked NMDAR-LTD, but not mGluR-LTD. NMDAR-LTD is associated with a decrease in the size of quantal excitatory postsynaptic currents, whereas for mGluR-LTD there was no change in quantal size, but a large decrease in the frequency of events. While mGluR-LTD did not interact with NMDAR-dependent long term potentiation (LTP), NMDAR-LTD was capable of reversing LTP. Prior saturation of mGluR-LTD had no effect on NMDAR-LTD. NMDAR-LTD and mGluR-LTD thus appear to be mechanistically distinct forms of synaptic plasticity in that they share neither induction nor expression mechanisms.

Quantal analysis of excitatory synapses in rat hippocampal CA1 in vitro during low-frequency depression.

1. We have performed a detailed quantal analysis of excitatory postsynaptic potentials (EPSPs) evoked by minimal extracellular stimulation in the CA1 region of slices of adult rat hippocampus maintained in vitro. 2. EPSPs were evoked at 2-5 Hz, and the eight that were analysed all showed at least a 50% depression of mean peak amplitude during recording. 3. EPSP amplitude fluctuations were analysed by three methods: the use of amplitude frequency histograms with clear and reliable peaks where available, graphs of the EPSP (coefficient of variation)-2 against EPSP mean, and analysis of EPSP mean and standard deviation assuming simple binomial statistics with the number of release sites (N) kept constant but the quantal size (Q) and the release probability (Pr) allowed to vary over time. 4. The results of the three analysis procedures were in good agreement. Seven EPSPs showed a substantial reduction in the mean number of quanta released per trial, and in three cases this was the predominant mechanism of the depression. Five EPSPs showed a substantial decrease in Q. Values for N ranged between 3 and 18, with a median of 6; Pr ranged between 0.14 and 0.81 and Q between 66 and 275 microV. 5. We used the Q estimates from the binomial method to correct the recorded EPSP amplitudes for changes in quantal size over time. For seven out of the eight EPSPs, this rescaling procedure allowed histograms with clear peaks to be obtained from longer runs of data, or improved the sharpness of the peaks in histograms from all the recorded data. The improvement in peak sharpness was assessed using an autocorrelation-based method. The correction was much less successful if the Q estimates were obtained with a variant of the binomial method in which Pr was held constant and N was allowed to vary. 6. The only simple explanation for the success of the correction procedure is that changes in quantal size were a major factor in obscuring peaks in histograms based on large numbers of trials, and that the quantal size estimates from the binomial method with N held constant were reasonably accurate. 7. We conclude that transmission at these synapses was quantal with relatively low quantal variance, but repetitive stimulation often induced substantial changes in the quantal parameters that might prevent the success of conventional quantal analysis approaches.

Two Distinct Forms of Long-Term Depression Coexist in CA1 Hippocampal Pyramidal Cells

Two distinct forms of long-term depression (LTD), one dependent on the activation of NMDA receptors (NMDARs) and the other dependent on the activation of metabotropic glutamate receptors (mGluRs), are shown to coexist in CA1 hippocampal pyramidal cells of juvenile (11–35 day-old) rats. Both forms were pathway specific and required membrane depolarization and a rise in postsynaptic Ca 2+. mGluR-LTD, but not NMDAR-LTD, required the activation of T-type Ca 2+ channels, group 1 mGluRs, and protein kinase C, while NMDAR-LTD, but not mGluR-LTD, required protein phosphatase activity. NMDAR-LTD was associated with a decrease in the size of quantal excitatory postsynaptic currents, whereas for mGluR-LTD there was no change in quantal size, but a large decrease in the frequency of events. NMDAR-LTD, but not mGluR-LTD, reversed NMDAR-dependent long-term potentiation, and NMDAR-LTD was unaffected by prior saturation of mGluR-LTD. These findings indicate that NMDAR-LTD and mGluR-LTD are mechanistically distinct forms of synaptic plasticity.

Nitric oxide modulation of quantal secretion in chick ciliary ganglia.

1. Long-term potentiation of quantal secretion was studied at ciliary ganglion synapses of post-hatched birds following tetanic stimulation of the oculomotor nerve and the effects of nitric oxide (NO) on quantal secretion were determined. 2. Tetanic stimulation of the oculomotor nerve at 30 Hz for 20 s at room temperature increased the amplitude of the excitatory postsynaptic potential (EPSP) by about 100%; 1-2 min after the tetanus the EPSP declined exponentially with a time constant of about 10 min (long-term potentiation; LTP). LTP was due to an increase in the quantal content of the EPSP not to a change in quantal size. 3. A component of LTP was shown to be due to the release of NO in the ganglion, as blocking the synthesis of NO with L-arginine methyl ester decreased the potentiation by 70%. 4. Exogenous application of NO using sodium nitroprusside increased the amplitude of the EPSP by more than 30% due to an increase in the quantal content of the EPSP. 5. Both 8-bromo-cGMP and 8-bromo-cAMP increased the quantal content of the EPSP by more than 44% without changing the quantal size. 6. The results suggest that endogenous NO is involved in either the initiation or maintenance phase of LTP. This may occur through an increase in quantal secretion consequent on the action of an elevated cGMP increasing cAMP.

Interaction between paired-pulse facilitation and long-term potentiation in area ca1 of guinea-pig hippocampal slices: Application of quantal analysis

The aim of the study was to further specify mechanisms of maintenance of hippocampal long-term potentiation. Previous analysis of excitatory postsynaptic potentials showed increases in quantal content (mean number of neurotransmitter quanta released by every testing pulse) with smaller increases in quantal size (effect of one transmitter quantum) following long-tenn potentiation induction. Here we recorded intracellularly excitatory postsynaptic potentials from CA1 pyramidal neurons of guinea-pig hippocampal slices after minimal paired-pulse stimulation of monosynaptic inputs. Statistical parameters underlying excitatory postsynaptic potential fluctuations were estimated by a deconvolution procedure using a quantal model. The parameters of excitatory postsynaptic potentials following paired-pulse stimulation were studied before and after induction of long-term potentiation. Under both conditions, paired-pulse facilitation was found to be accompanied by increases in quantal content and quantal size. During long-term potentiation, paired-pulse facilitation of amplitude and quantal content was lower. The respective changes in the paired-pulse facilitation ratios correlated with long-term potentiation magnitude. In contrast, the paired-pulse facilitation of quantal size did not change significantly following long-term potentiation induction. The results are compatible with the existence of two separate mechanisms of long-term potentiation maintenance. They support the suggestion that changes in quantal content are mainly due to presynaptic mechanisms which are shared by long-term potentiation and paired-pulse facilitation. The mechanisms underlying changes in quantal size are of a different nature for long-term potentiation and paired-pulse facilitation. For long-term potentiation they might be located postsynaptically.

Further evidence for the dynamic formation of transmitter quanta at the neuromuscular junction.

Fatt and Katz (Nature 166:597-598, 1950; J Physiol 117:109-128, 1952) attributed miniature endplate potentials (MEPPs) to the action of a standard quantity of transmitter, the quantum (Del Castillo and Katz, J Physiol 124:560-573, 1954). Quantal packets of transmitter were proposed to be preformed (Del Castillo and Katz, In CNRS Paris (Ed): "Microphysiologie comparée des éléments excitables" 67:245-258, 1957) and stored in large numbers in the motor nerve terminal. Statistical analyses of intervals between MEPPs and numbers of quanta composing small endplate potentials indicated that quantal release was a random process and that release sites functioned independently of each other. With the discovery of synaptic vesicles it was proposed that each contained one quantum of transmitter. The quantal-vesicular hypothesis (Del Castillo and Katz, as cited above) fails, however, to explain amplitude distributions of MEPPs that are skewed and/or that show multiple peaks (Kriebel et al., Brain Res Review 15:167-178, 1990). The drop formation process (Shaw, "The Dripping Faucet as a Model Chaotic System," Santa Cruz, CA: Aerial Press, Inc., 1984) was shown to generate amplitude classes of drops that were similar to classes of MEPPs which suggested that rapid changes in quantal size and ratios of skew- to bell-MEPPs could be explained with a simple dynamic process which determines quantal size at the moment of release (Kriebel et al., as cited above, 1990). Further similarities between miniature endplate currents (MEPCs) and the formation of drops are reported here. We found that rapid changes in MEPC amplitudes and time courses, which accompany an increase in frequency, mimic changes in drop sizes that accompany increases in flow rate. MEPC intervals have a minimum and their distributions are comparable to those of drop intervals. During an increased rate of transmitter release, MEPP amplitudes and intervals were positively correlated. The results suggest that spontaneously released transmitter "packets" are formed at the moment of release and that transmitter supply to the process that forms packets is continuous.

The effects of l-vesamicol, an inhibitor of vesicular acetylcholine uptake, on two populations of miniature endplate currents at the snake neuromuscular junction

The actions of the active l-isomer of vesamicol, an inhibitor of the vesicular storage of acetylcholine, has been studied on spontaneous and evoked acetylcholine release at the snake neuro-muscular junction. Miniature endplate currents and endplate currents were recorded from cut muscle fibres of the garter snake, Thamnophis sirtalis. In controls, prolonged periods of high frequency nerve stimulation produced a bimodal distribution of miniature endplate current amplitudes. The stimulation induced “small-mode” miniature endplate currents had a mean amplitude of around 40–55% of the pre-stimulation miniature endplate current. Relative to the normal-sized post-stimulation miniature endplate current, the proportion and, to a lesser extent, amplitude of the small-mode miniature endplate currents was related to both the frequency and duration of nerve stimulation and to the extracellular calcium ion concentration. In unstimulated preparations, l-vesamicol (2–5 μM) did not affect either endplate current quantal content or miniature endplate current amplitude or frequency. However, at these doses, the mean amplitude of the stimulation-induced, small-mode miniature endplate current was reduced by l-vesamicol in a concentration-dependent manner such that they were not visible at the highest dose. l-Vesamicol had no affect on the mean or coefficient of variance of amplitude of the larger, normal-sized miniature endplate current. Additionally, the stimulation-induced increase in overall miniature endplate current frequency seen in controls was abolished by 5 μM l-vesamicol. After prolonged 10 Hz nerve stimulation endplate current amplitude was markedly reduced in both controls (by 94%) and in the presence of 5 μM l-vesamicol (by 98%). Analysis of endplate current amplitude variance showed that in control the decrease was due to reductions in both quantal content and quantal size while in l-vesamicol the decrease was due entirely to a change in quantal content with no change in quantal size. Thus, we have observed that l-vesamicol selectively reduces the amplitude of a population of stimulation-induced small-mode quanta both as miniature endplate currents and as constituents of endplate currents. We suggest that these quanta are derived from a highly active, readily releasable pool. An action of l-vesamicol on this labile pool is consistent with previous observations on its ability to inhibit the vesicular storage of acetylcholine.

Reduction by baclofen of monosynaptic EPSPs in lumbosacral motoneurones of the anaesthetized cat.

1. Monosynaptic excitatory postsynaptic potentials (EPSPs) were elicited in lumbosacral motoneurones of pentobarbitone anaesthetized cats by stimulating group Ia muscle afferents with most of the dorsal roots severed. In some experiments Ia EPSPs were recorded together with monosynaptic EPSPs elicited by stimulating the ipsilateral ventral quadrants (VQ) of the thoracic spinal cord. Injection of (+/-) baclofen (1 mg kg-1 I.V.) caused a reduction in the peak amplitudes of both Ia and VQ EPSPs, which started immediately upon injection and progressed gradually. No recovery in EPSP amplitude was seen during the recording period, which lasted up to 60 min. 2. The Ia EPSP peak amplitude was reduced by 18-61% (mean +/- S.D., 38 +/- 14%; n = 30), while VQ EPSPs were reduced by 7-42% (23 +/- 13%; n = 5). Baclofen had a significantly larger effect on Ia EPSPs than VQ EPSPs (P less than 0.001; t test). 3. Baclofen did not cause any consistent change in the membrane potential, nor in the membrane time constant, as estimated from the exponential decay of the tail of the EPSP. There was no tendency for the reduction in peak EPSP amplitude to be related to the estimated electrical distance on the dendritic tree at which the synaptic current was injected. 4. For two I a and two VQ EPSPs, the trial-to-trial fluctuation in the peak amplitude was resolved into quantal parameters before and after baclofen was administered. The reduction in peak amplitude was in all cases accounted for by a reduction in the probability of release of neurotransmitter, with no change in quantal size. Other EPSPs either showed negligible trial-to-trial amplitude fluctuation, or could not be resolved into quantal parameters without ambiguity. 5. By comparing the variance components of the EPSP peak amplitude distribution, the hypothesis was tested that the entire action of baclofen was to reduce quantal amplitude. This was rejected for sixteen out of thirty Ia and three out of five VQ EPSPs (P less than 0.05). 6. These results support a presynaptic site of action of baclofen on the terminals of Ia afferents, by decreasing the probability of release of neurotransmitter. They also indicate a similar, although weaker, action on VQ terminals. No evidence was found for an action on the postsynaptic membrane properties or synaptic conductance.

Presynaptic and postsynaptic effects of histamine and histamine agonists in the superior cervical ganglion of the rat

Extracellular and intracellular recording techniques were used to study the effects of histamine and the histamine agonists [impromidine (IMP) and 2-thiazolylethyla Mine (2-TH)] on synaptic transmission in the superior cervical ganglion of the rat in vitro. At the concentrations employed (up to 10 −5 M) these compounds did not produce detectable effects on the electrical properties of the postsynaptic neurons. Histamine produced a dose-dependent reduction in the amplitude of the extracellularly-recorded presynaptic and postsynaptic compound action potential. The H 2 receptor agonist impromidine reduced only the postganglionic compound action potential. Cimetidine, a specific H 2 receptor antagonist, produced parallel shifts in the log dose-response curves for impromidine. Impromidine also reduced the average size of the evoked excitatory postsynaptic potential. The reduction of the mean amplitude of the excitatory postsynaptic potential was due to a decrease in the amount of acetylcholine (ACh) liberated by each preganglionic volley (mean quantal content, m) and a diminution in quantal size. The H 1 receptor agonist, 2-TH produced a dose-dependent increase in the presynaptic and postsynaptic compound action potential and in m. The increase in m was not associated with changes in quantal size. The H 1 antagonists, pyrilamine and promethazine, did not prevent facilitation of ganglionic transmission induced by 2-TH. It is concluded that histamine H 1 and H 2 receptors exist on preganglionic axons, or terminals in sympathetic ganglia of the rat. Activation of H 1 receptors facilitates release of ACh whereas H 2 receptor activation results in depressed release.

Effect of colchicine and vinblastine on crayfish neuromuscular junction

1. 1. The effect of the microtubule disrupting agents colchicine and vinblastine was studied on crayfish excitatory neuromuscular transmission. 2. 2. Colchicine, in concentrations of 1–3 mM, brought about a decrease in the amplitudes of intracellularly recorded excitatory junctional potentials (ejps). Vinblastine, in concentrations of 0.08–0.32 mM, increased the amplitudes of ejps. 3. 3. Drug-induced alterations in ejp amplitude resulted from changes in quantal content of the excitatory nerve as determined by extracellular recording. The effect could not be explained by changes in quantal size. 4. 4. The effect of colchicine and vinblastine does not appear to be related to an action on microtubules.